Main Outcome Measures The primary outcome measure was change on the Clinical Global Impression
of Change (CGIC) 7-point scale, analyzed by intent to treat; secondary outcome
measures included other global measures as well as measures of mood, specific
cognitive domains (memory, attention, and language), motor function, and activities
of daily living; compared by the combined estrogen groups vs the placebo group
at 2, 6, 12, and 15 months of follow-up.

Conclusions Estrogen replacement therapy for 1 year did not slow disease progression
nor did it improve global, cognitive, or functional outcomes in women with
mild to moderate AD. The study does not support the role of estrogen for the
treatment of this disease. The potential role of estrogen in the prevention
of AD, however, requires further research.

Figures in this Article

Alzheimer disease (AD) affects more than 4 million Americans and is
one of the most frequent obstacles to healthy aging in this country. Women
appear to be at higher risk for developing AD, only in part due to increased
longevity.1 Because women with AD also live
longer than men with AD, there are approximately twice as many women as men
in the population with this disorder. It has been suggested that the abrupt
decline of estrogen production in postmenopausal women may be associated with
a vulnerability of women to develop AD. Men, in contrast, have an intrinsic
supply of estrogen by having the ability to aromatize testosterone into estrogen
in the brain.

Considerable evidence has emerged from neuropathological studies,2- 4 animal behavioral studies,5,6 and human investigations7,8
to suggest that estrogen may be beneficial in improving cognition and mood
in AD. Several open-label clinical trials9- 11
and 1 randomized clinical trial12 reported
selective cognitive improvement in women with dementia who received estrogen
replacement therapy (ERT). However, these studies have all been relatively
brief, generally ranging from 6 to 8 weeks of treatment with estrogen. Similarly,
the number of subjects taking the drug in these trials has been small, ranging
from 7 to 12, and most have not used standardized diagnostic criteria. Thus,
the evidence for using estrogen as treatment for clinically diagnosed AD is
modest at best. Since the potential role of estrogen for the treatment of
AD is of public health significance, this intervention, if scientifically
supported, could become a routine part of the management regimen for women
with AD.

We conducted this study to provide a definitive, placebo-controlled,
double-blind, randomized clinical trial of adequate duration and size to determine
the benefit of ERT for the treatment of women with mild to moderate AD. We
chose to study unopposed estrogens because previous investigations have suggested
that progesterone may mitigate some of estrogen's beneficial effects in the
central nervous system.

METHODS

Objectives

The specific aims of the study were to (1) determine if women with AD
treated with unopposed estrogens would experience improvement or stability
in cognition and other parameters during or after 12 months of therapy; (2)
determine which components of the psychometric assessment were improved or
stabilized by treatment; (3) determine whether there was a differential response
to 2 dosages of estrogen; and (4) establish the safety and tolerability of
estrogen in elderly women with AD.

Participants

Participants for this study were recruited from participating sites
of the Alzheimer's Disease Cooperative Study (ADCS; a consortium supported
by the National Institute on Aging), with enrollment of 120 women with hysterectomies
between October 1995 and January 1999. Selection of women with hysterectomies
allowed for the subjects' long-term exposure (1 year) to unopposed estrogen
therapy while eliminating the risk and safety concern of endometrial hyperplasia,
which occurs in prolonged unopposed estrogen administration in women with
an intact uterus. Thirty-two sites (designated as AD centers by the National
Institute on Aging and/or selected sites of the ADCS) participated in the
recruitment of these subjects. General inclusion criteria included a diagnosis
of probable AD according to National Institute of Neurological and Communicative
Disorders and Stroke–Alzheimer's Disease and Related Disorders Association
criteria13 in the mild to moderate stage (the
study protocol specified a Mini-Mental State Examination [MMSE]14
score of 14-28; several exceptions were made by the project director to allow
for participants with MMSE scores as low as 12); female sex; previous hysterectomy
(oophorectomy not required); age older than 60 years; absence of major clinical
depressive disorder (as measured by scores of <17 on the Hamilton Depression
Rating Scale [Ham-D]15); and normal gynecological,
breast, and mammography examination results.

Exclusion criteria were myocardial infarction within 1 year, history
of thromboembolic disease or hypercoagulable state, hyperlipidemia, or use
of excluded medications (ie, estrogens within 3 months; current use of antipsychotics,
anticonvulsants, anticoagulants, β-blockers, narcotics, methyldopa, clonidine,
or prescription cognitive-enhancing or antiparkinson medications, including
experimental medications within 60 days prior to baseline. Stable dosages
of neuroleptics, antidepressants, anxiolytics, sedatives, and hypnotics were
allowed). At the initiation of the protocol, individuals treated with donepezil
or tacrine were excluded, but a protocol amendment after 20 months of enrollment
allowed the stable use (minimum of 4 weeks) of these medications before screening
for the study.

Intervention

This study used a 12-month, randomized, double-blind, placebo-controlled,
parallel-group design, in which 120 women were enrolled and randomized to
receive a single daily dose of placebo, Premarin (conjugated equine estrogens
[CEEs], Wyeth-Ayerst Pharmaceuticals, St Davids, Pa), 0.625 mg, or Premarin,
1.25 mg, followed by a 3-month, single-blind placebo washout phase for all
women. Conjugated equine estrogens were chosen for this trial because this
formulation is the most commonly prescribed form of ERT. Additionally, CEEs
include multiple components, some of which have been shown to have neurotrophic
properties, which could be beneficial to brain function.

Participants were randomly allocated to 1 of the 3 treatment arms (Figure 1). Treatments were assigned in randomized
permuted blocks of 6 and shipped to each site at the start of the study. The
ADCS biostatistical division generated and archived the randomization list.
Boxes containing 7-day blister cards of study medication were packaged and
shipped by the manufacturer according to instructions provided by the ADCS.
Patients were instructed to take 2 identically appearing tablets of study
medication each morning. Subjects assigned to estrogen, 1.25 mg/d, received
2 estrogen 0.625-mg tablets; those assigned to estrogen, 0.625 mg/d, received
1 placebo tablet and 1 estrogen 0.625-mg tablet; those assigned to placebo
received 2 placebo tablets. Hereafter, the 0.625-mg/d and 1.25-mg/d estrogen
dosages are referred to as low and high dosages, respectively. All subjects
in this study were required to have a caregiver who administered the investigational
agent during the trial. Compliance monitoring was done through plasma estradiol
level evaluation at each visit and pill count.

Figure 1. Participant Flow Diagram

Cognitive, global, and other outcome measures were evaluated at screening,
baseline, and 2, 6, 12, and 15 months. A telephone check was performed at
the 4-month interval to verify ongoing administration of the experimental
medication and the status of concurrent medications and to address any issues
or concerns, and a brief safety visit was conducted at the 9-month interval.
The study was reviewed and approved by the institutional review board at each
site. Written informed consent was obtained from all participants.

Outcome Measures

The ADCS version of the Clinical Global Impression of Change (CGIC)
scale, developed as a semistructured interview from the traditional CGIC scale,16 was the primary outcome measure used to assess change
from baseline. On this scale, scores of 1, 2, and 3 represent marked, moderate,
and mild improvement, respectively; 4 represents no change; and 5, 6, and
7 represent mild, moderate, and marked worsening, respectively.

The MMSE (range, 0-30) and the Clinical Dementia Rating Scale17 (CDR; range, 0-5) also were used as global staging
instruments. Through the judicious choice of other secondary outcome measures,
this study also aimed to address several unanswered questions: (1) independent
of mood enhancement, does estrogen therapy improve cognition in AD; (2) by
what mechanism is memory improved; and (3) what other clinically relevant
benefit does estrogen have in AD?

The first goal was to determine to what degree observed cognitive benefit
was associated with improved mood. To this end, it was proposed that the evaluation
of estrogen include a traditional measure of depression (Ham-D) and an index
of mood state (Multiple Affect Adjective Checklist–Revised [MAACL-R]18) conducted concurrently with other cognitive assessments.
To elucidate possible mechanisms for estrogen's effect on memory that are
separate from specific mood alteration, the assessment of memory included
measurement of (1) explicit verbal learning (Alzheimer's Disease Assessment
Scale–Cognitive [ADAS-Cog]19), (2) mood-congruent
memory (Emotional Face Recognition Test; unpublished data, Elizabeth Koss,
PhD, 1995), and (3) visual delayed nonmatched to sample recognition (New Dot
Test20). We investigated other cognitive benefits
that could be associated with estrogen by testing the subjects on measures
of attention (Letter Cancellation,21 Trail-Making
Test A,22 and Digit Symbol23),
language (Category Fluency24 and Letter Fluency25), and motor behavior (Grooved Pegboard Test26 and Finger Tapping Test27).
Another goal in the choice of secondary measures was to assess the effect
of estrogen on activities of daily living abilities in AD, as measured by
the Blessed Dementia Rating Scale28 and the
Dependency Scale.29

Safety Monitoring

To reduce risk associated with estrogen administration, screening measures
before enrollment included a baseline gynecological and breast examination
within the 3 preceding months, a mammogram within the 6 preceding months,
and a Papanicolaou test within the previous 3 years. As an additional safety
measure, the mammogram was repeated at the end of the 12-month double-blind
phase of the study to monitor for any breast complications. At each visit,
blood pressure, body weight, and fluid retention (ankle swelling) were monitored.
An additional lipid profile was performed at the 2-month visit to detect the
complication of hyperlipidemia as a rare reaction to estrogen compounds. Adverse
event reports were reviewed quarterly by the independent ADCS safety monitoring
committee, who found no necessity to break the blind or interrupt the trial
at any time.

Patient Populations

For purposes of analysis, 3 patient populations were defined. These
included traditional intent-to-treat (based on a last observation carried
forward imputation scheme) and completers populations (12-month visit completed)
as well as a compliers population (defined as all individuals who completed
the study and ingested at least 80% of the randomized agent by pill count).
Of the 120 randomized subjects, 119 were exposed to the investigational agent
(1 subject dropped out because of medical problems before starting the medication). Figure 1 shows the subject flow and disposition
through the course of the trial. Of the 120 randomized subjects, 97 completed
the trial. There was no attempt to balance the use of donepezil across treatment
groups. Table 1 shows that more
patients in the ERT groups took donepezil during the course of the trial compared
with the placebo group.

Statistical Analyses

Power calculations were performed using data from a clinical trial with
a similar design that included only women aged at least 60 years with baseline
MMSE scores of 16 to 28. Based on the data from this similar trial, with 40
subjects receiving placebo and 80 subjects receiving estrogen, the design
power was 81% to detect a 29% difference in the proportion of subjects who
worsen in the 2 groups (60% worse in the placebo group vs 31% worse in the
estrogen group) using a 2-tailed α = .05. Since this was a dosage-finding
study, a large effect size was sought to ensure clinical meaningfulness and
to estimate the signal size for a possible follow-up trial, if the findings
were positive.

In all analyses, a set of predefined covariates was assessed as potential
confounders (subject age, apolipoprotein e4 allele frequency, subject education).
Any variables unbalanced at baseline (P≤.15) and
significantly associated with response (P≤.10)
were included in the statistical model. In the 2-group analysis (combined
estrogen groups vs placebo group), there was no significant imbalance of the
covariates at baseline, negating the inclusion of these prestated potential
confounders to the statistical models. In the 3-group analysis (differential
dosage response vs placebo), subject age at baseline was marginally unbalanced
(P = .07) in the low-dosage estrogen group, and was
significantly associated with 3 outcome variables. Therefore, age was included
as a covariate in the statistical analysis models for the ADAS-Cog (P = .07), Dependency Scale (P
= .07), and Grooved Pegboard (P = .06).

The primary end point used to evaluate the differential effect of estrogen
on progression of AD was the ADCS-CGIC. For this analysis, the 7-point scale
was collapsed to 5 points because of lack of subjects in the marked or moderately
improved categories and was analyzed using ordinal logistic regression. Other
primary and secondary end-point treatment differences were analyzed as follows:
for continuous measures, linear regression adjusting for baseline values (analysis
of covariance) was applied. For categorical measures, logistic regression
adjusting for baseline values was used. Confirmatory analyses were conducted
using linear regression on changes in scores with no baseline adjustment and χ2 tests (with Mantel-Haenszel adjustment if necessary) for categorical
measures. No interim analyses were performed.

RESULTS

The demographic and clinical characteristics of each group at baseline
are illustrated in Table 1. No
significant group differences were seen on any characteristic at baseline
(all P values >.05).

Two-Group Comparison

To assess the overall efficacy of estrogen, the low- and high-dosage
estrogen groups were combined into a single group of 81 women with AD and
hysterectomies who took estrogen. These 81 women were compared with the 39
placebo subjects regarding performance on the ADCS-CGIC, MMSE, ADAS-Cog, and
the CDR at 12 months to assess change. The primary intent-to-treat analysis
comparing the combined estrogen groups with the placebo group showed no difference
between groups for the percentage of patients who worsened on the ADCS-CGIC
(P = .48), the ADCS-CGIC score (P = .43), the MMSE score (P = .51), or the
ADAS-Cog score (P = .13). However, a significant
difference was seen on the CDR (P = .01) favoring
the placebo group (Table 2 and Figure 2). Repeated completers and compliers
analyses on the primary outcome measure likewise did not reveal any differential
treatment effects on the ADCS-CGIC (data available from the authors on request).

Analysis of secondary outcomes with sensitivity to changes in mood (Ham-D),
memory (Emotional Face Recognition Test and New Dot Test), attention (Letter
Cancellation, Trail-Making Test A, and Digit Symbol) and activities of daily
living (Blessed Dementia Rating Scale and Dependency Scale) showed no significant
differences between treatment and placebo groups (Table 2). Among the language measures, Category Fluency favored
the placebo group (P = .05), yet there were no group
differences on Letter Fluency. Of the 2 motor measures, the Grooved Pegboard
Test did not detect group differences, but the Finger Tapping Test favored
the placebo group (P = .05). Similar analyses on
completer and complier populations showed consistent results (data available
on request).

Three-Group Comparison

A separate analysis addressed treatment effect differences between each
of the 2 dosages of estrogen compared with placebo. Again, the 12-month intent-to-treat
analysis showed no difference between groups for the proportion who worsened
on the ADCS-CGIC (P = .73 for both low and high dosages),
the ADCS-CGIC score (low dosage, P = .66; high dosage, P = .36), the MMSE score (low dosage, P = .48; high dosage, P = .64), or the ADAS-Cog
score (low dosage, P = .09; high dosage, P = .32). However, a significant difference was seen on the CDR at
12 months (low dosage, P = .03; high dosage, P = .01), again favoring the placebo group (Table 3 and Figure 3).
In addition, we found a benefit of low-dosage estrogen on the MMSE change
in score after 2 months of exposure (low dosage = −0.36; placebo = −1.64; P = .05), but the benefit did not persist with continued
treatment. There was no evidence of improvement in global functioning at any
point in the trial. Repeated completers and compliers analyses on the primary
outcome measure likewise did not reveal any differential treatment effects
on the ADCS-CGIC (data available from the authors on request).

Figure 3. Three-Group Intent-to-Treat Analysis

Data are the mean changes from baseline for the Alzheimer's Disease
Cooperative Study version of the cognitive tests (see legend to Figure 2 for
explanation of abbreviations). Plotted values for the CDR scores are computed
to 2 decimal places. Error bars represent 95% confidence intervals around
the mean values; gray lines indicate baseline. See footnote to Table 1 for explanation of treatment groups.

Analysis of the remaining secondary outcome variables showed either
nonsignificant differences between groups or results that favored the placebo
group (Table 3). The Ham-D, a
measure of mood, did not differ between groups. The subscale factors of the
MAACL-R also did not differ between groups (data available from the authors
on request). There were no detectable group differences in memory, attention,
or language measures. Among the motor measures, the Grooved Pegboard Test
did not detect group differences, but the Finger Tapping Test again favored
placebo, but only in the low-dosage group (P = .04).
Measures of activities of daily living were not significantly different between
groups. Repeated analyses on completer and complier populations showed the
same results (data available from the authors).

Two additional analyses were performed to compare results on the primary
outcome measures among women with prior estrogen exposure and then among women
with prior donepezil treatment. Within each treatment group (placebo, low-dosage
and high-dosage estrogen), the participants with a history of estrogen use
were compared with the participants who had no history of estrogen use prior
to the present trial on four 12-month outcome variables, the CGIC, CDR, MMSE
and ADAS). As reflected in Table 1,
years of prior estrogen exposure were comparable between the placebo and high-dosage
estrogen groups (5.4 vs 6.4 mean years). For the placebo group, mean differences
on the outcome measures were not significant for prior estrogen use vs nonuse,
respectively, (CGIC, 5.0 vs 5.1, P = .32; CDR, 1.3
vs 1.2, P = .42; MMSE, 18.3 vs 17.5, P = .90;ADAS-Cog, 27.9 vs 25.8, P = .41).
For the high-dosage group, mean differences were likewise not significant
(CGIC, 5.4 vs 5.0, P = .07; CDR, 1.7 vs 1.5, P = .24;MMSE, 16.0 vs 19.4, P
= .21; ADAS-Cog, 31.4 vs 26.2, P = .97). However,
participants in the low-dosage estrogen group with a longer period of prior
estrogen exposure (16.8 mean years) had significantly better mean CGIC scores
at 12 months (4.8 vs 5.5; P = .04), but no significant
impact on the CDR (mean, 1.4 vs 1.8; P = .71), the
MMSE (mean, 18.8 vs 16.4; P = .61), and the ADAS-Cog
(mean, 26.6 vs 34.1; P = .86). A similar analysis
of donepezil users vs nonusers showed no significant differences in performance
on the CGIC, CDR, MMSE, or the ADAS-Cog outcome variables.

Treatment-emergent adverse events, grouped categorically, were not significantly
different between placebo and estrogen-treated groups. However, 2 clinically
important issues arose during the trial: 4 episodes of vaginal bleeding occurred,
representing protocol violations because these 4 women had not had prior hysterectomies,
despite confirmatory gynecological examinations before randomization into
the protocol; and 4 episodes of deep vein thrombosis occurred, 2 in the low-dosage
and 2 in the high-dosage estrogen groups. Two patients died, 1 in each of
the estrogen groups. Both deaths were sudden but neither death was believed
to be related to treatment medication.

COMMENT

Estrogen failed to improve cognitive or functional outcomes in this
1-year study of women with mild to moderate AD and hysterectomies. Similar
to previous reports, we found a benefit of low-dosage estrogen on the MMSE
after brief exposure (2 months; P = .05), but the
benefit did not persist with continued treatment. In fact, patients receiving
estrogen appeared to decline more than those receiving placebo on 1 global
clinical measure, the CDR, despite the greater use of donepezil in the estrogen-treated
patients. Overall, the results of this study do not support the role of estrogen
in the treatment of AD.

To date, this study is the largest and the longest study to examine
estrogen as a treatment for women with AD. Given that patients receiving estrogen
did no better or worse than patients receiving placebo, the use of a larger
sample size would not have changed this result.

There are several plausible explanations for the difference between
the results of this study and previous studies. Animal studies indicate that
in the neural tissue, estrogen modulates cholinergic,3
serotonergic,30 and catecholaminergic31 neurotransmitter systems; regulates synaptogenesis
during the estrous cycle; regulates neurogenesis32;
and is neuroprotective,33 reducing the brain
damage associated with ischemic insult.34,35
It is possible that short-term improvements that have been seen in some clinical
trials were due to gene-dependent regulation of neurotransmitter systems such
as the up-regulation of cholinergic activity.36
A second mechanism of estrogen action could involve surface receptor–associated
signaling via ion channels, modulating electrical properties of neurons and
transmitter release processes.37,38
Such mechanisms are palliative, however, and insufficient to prevent decline
over the long term.

In basic studies on the mechanisms underlying neurodegeneration, it
has been suggested that there are at least 2 phases in the process, an initiation
phase and a propagation phase.39 It is hypothesized
that estrogen can delay the initiation phase but is insufficient to slow the
propagation phase. Thus, for example, cell culture studies on primary hippocampal
neurons show that estrogen is only partially protective against a variety
of insults.33 Estrogen appears to operate in
part through a gene-dependent up-regulation of antiapoptotic proteins in the
bcl-2 family in vitro and in vivo.33,34
In the AD brain, these genes are already up-regulated and, thus, further benefit
may not be gained.40 Estrogen also has antioxidant
properties, though they are relatively weak compared with vitamin E. The present
data suggest that the antioxidant capacity of estrogen is evidently insufficient
to slow progression. In addition, the anatomical organization of estrogen
receptors may favor a role in early stages. Estrogen receptors are most concentrated
in brain regions involved with the initial stages of the disease (eg, the
limbic system). As degeneration spreads to other regions, estrogen might be
unable to regulate gene-dependent defense mechanisms. Other mechanisms also
show selectivity for the initiation and propagation phases. For example, apolipoprotein
e4 appears to accelerate disease onset, but most studies agree that it does
not slow the rate of progression,39,41,42
although there have been exceptions.43 While
the mechanisms underlying the present results are as yet unknown, the data
suggest that some therapeutic interventions may only act during selective
phases of the disease process.

Thus, in the intact healthy brain, estrogen could play a key neuroprotective
role by delaying the initiation phase of neurodegenerative disease onset,
thereby supporting the finding of reduced risk of dementia from several published
epidemiological studies.44- 47
Two multicenter prevention trials are currently under way to answer this question
prospectively.

Of public health concern is the tendency for experimental treatments
to become standard of care before the rigorous scientific evidence is thoroughly
gathered. Such is the concern with estrogen administration for women with
AD. Numerous publications with broad-based distribution48,49
are now supporting the addition of ERT to the armamentarium of treatments
for women with AD as a means to enhance cognitive function and delay progression
of the disease. Such clinical practice, begun in advance of rigorous clinical
trials, could prove to be detrimental to patient outcome. While other ongoing
investigations (Women's Health Initiative–Memory Study, Women's International
Study of Long Duration Oestrogen for Menopause, and Preventing Postmenopausal
Memory Loss and Alzheimer's with Replacement Estrogens study) will provide
needed data on hormone replacement therapy in the primary prevention of AD,
this study does not support the role of estrogen for treatment of established
AD. However, there remains a possibility that estrogen could have an important
role as an adjuvant treatment, or as a means of delaying onset of disease.
Further investigation of ERT in these areas is still warranted.

Figure 3. Three-Group Intent-to-Treat Analysis

Data are the mean changes from baseline for the Alzheimer's Disease
Cooperative Study version of the cognitive tests (see legend to Figure 2 for
explanation of abbreviations). Plotted values for the CDR scores are computed
to 2 decimal places. Error bars represent 95% confidence intervals around
the mean values; gray lines indicate baseline. See footnote to Table 1 for explanation of treatment groups.

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